The enzymes of the catechol and homoprotocatechuate (hpc) meta-fission pathways are rich in mechanistic, structural, and evolutionary questions. The long-term goal of this research is to answer these questions by a combination of mechanistic enzymology, molecular biology, and x-ray crystallography. Significant progress has been made so that fundamental questions about the origin of the pathways and the evolution of the enzymes in them can be addressed, making them model systems for how catabolic pathways are assembled. These studies will also enhance our understanding of enzyme mechanisms, enol and dienol chemistry, and may assist in bioremediation efforts, design of biocatalysts, and lead to a better understanding of how medically relevant enzymes (e.g., beta-lactamases) evolved. During the last funding period, we identified members of the 4-oxalocrotonate tautomerase (4-OT) family, one of the families in the tautomerase superfamily, and determined their defining characteristics. Nature has apparently used the beta-alpha-beta- scaffold, the key building block for tautomerase superfamily members, as a template to make several new enzymes. 4-OT, an enzyme in the catechol pathway and the title member of the family, retains these activities as low-level ones. The structural basis for these activities will be determined and, with this information in hand, the activities amplified. A fumarylacetoacetate hydrolase (FAH)-like superfamily has now been identified and consists of the decarboxylases in the catechol and hpc pathways and an E. coli homologue designated YcgM. The signature motif and characteristics of the FAH-like superfamily will be determined. The major specific aims will be to: 1) determine the structural basis for the trans-3-haloacrylate dehalogenase activity of 4-OT using mutagenesis, stereochemical probes, and crystallography; 2) use rational and random mutagenesis to amplify 4-OT's dehalogenase activity; 3) characterize the cis-3-haloacrylate dehalogenase and decarboxylase activities of 4-OT; 4) characterize the decarboxylase and tautomerase domains of COHED and the presumed decarboxylase activity of YcgM using mutagenesis, inhibitors, and crystallography; 5) synthesize epoxide inhibitors of vinylpyruvate hydratase and obtain a crystal structure in order to discover its superfamily.